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Previous studies in rapidly proliferating rodent cells have suggested that the lethal effect of the DNA topoisomerase I inhibitor, camptothecin (CPT) is dependent upon the active participation of DNA replication (Holm et al. Cancer Res. 49:6365-6368; 1989). The purpose of the current study was to determine if this relationship applies to more slowly growing human cells. In our present study, we employed the human colon carcinoma cell line, HT-29 (45 hr doubling time). Flow cytometric determination of S-phase cells either by S-phase fit model or rectangle fit model analysis predicted that 21% of exponentially growing HT-29 cells were undergoing DNA replication. These findings were confirmed by immunofluorescence microscopy of bromodeoxyuridine labeled cells. Based on these findings, we would have expected only 20-30% of the cells to be susceptible to brief treatment (30 min) with CPT. Instead, 90-95% of HT-29 cells were killed. This apparent disparity was not due to prolonged cellular retention of drug after treatment because protein-linked DNA strand breaks reversed within 15 min of drug removal. Moreover, the DNA replication inhibitor, aphidicolin, fully protected HT-29 cells against CPT-induced killing but did not affect the production of CPT-induced protein-linked DNA strand breaks. Similar results were also obtained using the CPT-analog, 10,11-methylenedioxy-camptothecin, which was 5- to 10-fold more potent than camptothecin (O'Connor et al. Cancer Commun. 2:395-400; 1990).(ABSTRACT TRUNCATED AT 250 WORDS)

The kinetics of formation and removal of DNA interstrand crosslinks (ISC), DNA-protein crosslinks (DPC), and single strand breaks (SSB) by several nitrogen mustards were compared in order to determine the degree to which lesion selectivity may vary. The kinetic measurements using DNA alkaline elution methodology were obtained in mouse L1210 cells treated with mechlorethamine (HN2), phenylalanine mustard (L-PAM), uracil mustard (UM), 6-methyl-UM, and quinacrine mustard (QM). The ISC or DPC challenge delivered to cells was gauged on the basis of the kinetics as either total ISC or DPC produced, or as the area under the lesions versus time curve (AUC). By either measure (excepting QM), ISC correlated well with loss of colony survival, whereas DPC did not. The ISC/DPC ratio may therefore be a useful index of lesion selectivity. This ratio was significantly greater for 6-methyl-UM than for HN2. The ratio was also greater for L-PAM than for HN2 but only when gauged by AUC; this was attributable to an unusually slow rate for ISC removal in the case of L-PAM. The preferential reaction of UM at some 5'-GC-3' sites in purified DNA had suggested that UM might produce ISC with increased efficacy. UM, however, was somewhat less efficacious in ISC production than was 6-methyl-UM, which lacked selectivity for alkylation at 5'-GC-3'. QM was the only compound that produced detectable SSB, and the SSB were so numerous that ISC could not be quantitated.(ABSTRACT TRUNCATED AT 250 WORDS)

We had previously shown that 10,11-methylenedioxy-20-(RS)-camptothecin (MDO-CPT) is a more potent inhibitor of purified DNA topoisomerase I than 20-(S)-camptothecin (CPT). The current studies compared the cytotoxicity and DNA damage induced by MDO-CPT and CPT in the human colon carcinoma cell line, HT-29. MDO-CPT was 7- to 10-fold more potent than CPT both for cytotoxicity (ID50 = 25 vs. 180 nM) and production of DNA single-strand breaks (SSB). Kinetics of SSB formation and reversal were similar for MDO-CPT and CPT. DNA-protein crosslinks (DPC) were also produced by both drugs with a SSB/DPC ratio of 1/1. Moreover, no SSB were detected under non-deproteinizing conditions, indicating that both CPT and MDO-CPT produced protein-linked DNA single-strand breaks. A good correlation between cytotoxic potency and protein-linked DNA single-strand break production was observed for CPT and MDO-CPT, implying a causal relationship between drug-induced cytotoxicity and topoisomerase I inhibition. The sensitivity of human colon HT-29 cancer cells to camptothecins may be a selective phenomenon since these cells normally express natural resistance to current chemotherapeutic drugs, including topoisomerase II inhibitors.